Continuation is planned of a long-term study of plasmid molecular biology, focusing on the model rolling circle plasmid, pTl8l. Studies are proposed of the mechanisms of initiation and termination of leading strand replication, with special attention to the possible involvement of a type C cruciform structure containing the specific nick site. A comprehensive mutational analysis of the plasmid-coded initiator (Rep) protein is proposed, with the aim of isolating and localizing mutations affecting each of the protein's nine known functions. Attention will be devoted to the effects of local and distal secondary structures and other special sequence elements on the function of the pTl8l leading strand origin, with special reference to cis-inhibitory sequences. We have predicted and verified the replication-specific inactivation of the Rep protein and have demonstrated that the inactive form of the protein is modified. The nature of the modification and the mechanism of its formation will be determined.